A Lot More Advanced Biotechnology Tools

DNA Sequencing

– dideoxynucleotides• ddATP, ddGTP, ddTTP,

ddCTP• missing O for bonding of

nextnucleotide

• terminates the growing chain

DNA Sequencing

DNA Sequencing• Sanger method

– synthesize complementary DNA strand in vitro

– in each tube:• “normal” N-bases• dideoxy N-bases

–ddA, ddC, ddG, ddT

• DNA polymerase• primer• buffers & salt

2

1

3

4

2

Reading the sequence• Load gel with sequences from ddA,

ddT, ddC, ddG in separate lanes– read lanes manually & carefully– polyacrylamide gel

Fred Sanger 1978 | 1980

• This was his 2nd Nobel Prize!!– 1st was in 1958 for the

structure of insulin

The Sanger Method:

DNAi video

tutorial

Advancements in sequencing

• Fluorescent tagging– no more radioactivity– all 4 bases in 1 lane

• each base a different color• Automated reading

Applied Biosystems, Inc Applied Biosystems, Inc (ABI) built an industry on (ABI) built an industry on these machinesthese machines

More Advancements in sequencing

Capillary tube electrophoresisno more pouring gelshigher capacity & faster

PUBLICPUBLICJoint Genome Institute Joint Genome Institute

(DOE)(DOE)MITMITWashington University of Washington University of

St. LouisSt. LouisBaylor College of Baylor College of

MedicineMedicineSanger Center (UK)Sanger Center (UK)PRIVATEPRIVATECelera GenomicsCelera Genomics

Big labs!economy of scale

Automated Sequencing machines

Really BIG labs!

Human Genome Project• U.S government project

– begun in 1990• estimated to be a 15 year

project– DOE & NIH

• initiated by Jim Watson• led by Francis Collins

– goal was to sequence entire human genome

• 3 billion base pairs• Celera Genomics

– Craig Venter challenged gov’t – would do it faster, cheaper– private company

Different approaches

3. Assemble DNA sequence using overlapping sequences.

““map-based method”map-based method”gov’t methodgov’t method

““shotgun method”shotgun method”Craig Venter’s methodCraig Venter’s method

1. Cut DNA entire chromosome 1. Cut DNA entire chromosome into small fragments and clone.into small fragments and clone.

2. Sequence each segment & 2. Sequence each segment & arrange based on overlapping arrange based on overlapping nucleotide sequences.nucleotide sequences.

1.1. Cut DNA segment into fragments, Cut DNA segment into fragments, arrange based on overlapping arrange based on overlapping nucleotide sequences, and clone nucleotide sequences, and clone fragments.fragments.

2. Cut and clone into smaller fragments.2. Cut and clone into smaller fragments.

Shotgun!

Human Genome Project• On June 26, 2001, HGP published the “working

draft” of the DNA sequence of the human genome (4 years ahead of schedule).

• Historic Event! – blueprint

of a human– the potential to

change science & medicine

Sequence of 46 Human Chromosomes

3 billion base pairs

3G of data

Things Are Strange In Here:

Raw genome data

NCBI GenBank

• Database of genetic sequences gathered from research

• Publicly available on Web!

Maps of human genes…

• Where the genes are…– mapping genes & their mutant alleles

Defining a gene…• “Defining a gene is problematic because…

one gene can code for several protein products, some genes code only for RNA, two genes can overlap, and there are many other complications.”

• – Elizabeth Pennisi, Science 2003

gene

polypeptide 1polypeptide 1

polypeptide 2polypeptide 2

polypeptide 3polypeptide 3

proteinproteingene

RNARNAgene

 Types of DNA sequences in the human genome Exons (regions of genes coding

for protein, rRNA, tRNA) (1.5%)

RepetitiveDNA thatincludes

transposableelements

and relatedsequences

(44%)

Introns andregulatorysequences

(24%)

UniquenoncodingDNA (15%)

RepetitiveDNA

unrelated totransposable

elements(about 15%)

Alu elements(10%)

Simple sequenceDNA (3%)

Large-segmentduplications (5–6%)

And we didn’t stop there…

Genome Sizes and Estimated Numbers of Genes*

What have we found?• When you go looking…

…you will certainly find something!

Sing A Silly Song Extolling Automation!

Ethical Questions…

• 1. You have a familiar history of a terminal genetic disease. A genetic test exists for the disease. Would you take the test to see if you will have the disease?

•

• 2. As a condition of your continued employment, your boss wants you to have a genetic screening test and the results sent to the company. Will you get the test?

3. You and your spouse have decided to have a child. You have the resources to pre-determine your child’s gender. Would you do this?

4. What about your child’s intelligence?

• 5. You discover your favorite food contains genetically modified ingredients. Will you continue to eat the food?

6. As part of a routine medical procedure, your doctor discovers that you have a rare, beneficial variant of a protein that protects you from heart disease. Should your doctor be able to patent the protein?

7. Should you be entitled to any money from the patent?